Method of reducing neuronal cell damage

ABSTRACT

The present invention is directed to a method of reducing the occurrence of neuronal cell damage, including death, caused by transient cerebral hypoxia and/or ischemia. The method comprises the steps of: diagnosing a subject having a transient cerebral hypoxic and/or ischemic condition; and within 16 hours after onset of the condition, administering to the subject a neuroprotective amount of a pharmaceutical agent. The pharmaceutical agent is preferably selected from the group consisting of: a central nervous system stimulant (CNSS), monoamine neurotransmitter, monoamine oxidase inhibitor (MAOI), tricyclic antidepressant (TCA), or a combination thereof. Preferred agents include amphetamines, methamphetamine, methylphenidate, methylenedioxymethamphetamine, or a combination thereof.

RELATED APPLICATION DATA

This application claims the benefit of U.S. Provisional Application No.60/839,974 filed Aug. 23, 2006, the content of which is expresslyincorporated herein in its entirety by reference thereto.

TECHNICAL FIELD

The present invention is directed to a method of reducing the occurrenceof neuronal cell damage, including cell death, caused by transientcerebral hypoxia and/or ischemia. The method comprises the steps of:diagnosing a subject having a transient cerebral hypoxic and/or ischemiccondition and within 16 hours after onset of the condition,administering to the subject a neuroprotective amount of apharmaceutical agent. The pharmaceutical agent is preferably selectedfrom the group consisting of: a central nervous system stimulant (CNSS),monoamine neurotransmitter, monoamine oxidase inhibitor (MAOI),tricyclic antidepressant (TCA), or a combination thereof. Preferredagents include amphetamines, methamphetamine (MA), methylphenidate,methylenedioxymethamphetamine, or a combination thereof.

BACKGROUND OF THE INVENTION

Strokes are the leading cause of disability among adults, with over 80%involving ischemic insult. To date, no preventative or neuroprotectivetherapy has proven to be efficacious in humans. Amphetamines are one ofthe most extensively studied and promising group of drugs used tofacilitate stroke recovery after neuronal cell damage has occurred (see(Martinsson and Eksborg 2004)). In rats, a single dose of amphetamines(e.g., dexamphetamine) administered 24 hrs after sensorimotor cortexablation promotes hemiplegic recovery (Feeney et al. 1982). Thisbeneficial effect has been confirmed in a variety of different focalinjury models and species (Sutton et al. 1989; Hovda and Fenney 1984;Hovda and Feeney 1985; Schmanke et al. 1996; Dietrich et al. 1990;Stroemer et al. 1998). In each of these studies ischemic injury wasmodeled by the permanent ligation/embolism of a vascular component, orcortical ablation.

A different type of ischemic injury involves the transient interruptionand reperfusion of blood flow to the brain. The hippocampus is extremelysensitive to this type of ischemic insult. In humans and experimentalrodent models, brief ischemic episodes can result in the selective anddelayed death of neurons located in the hippocampus, especially thepyramidal cells of the CA1 sector (Kirino 1982). This type of lesionimpairs performance on cognitive tasks that involve spatial memory(Zola-Morgan et al. 1986; Squire and Zola-Morgan 1991). Althoughamphetamine administration is associated with improved behavioralrecovery in models of focal ischemia or cortical ablation, the prior artreported that treatment with amphetamines does not reduce infarct volumeand thus, is not a preventative or neuronal protectant. The prior artalso suggest that amphetamines facilitate behavioral recovery aftercortical injury by influencing brain plasticity (Gold et al. 1984) aswell as resolution of diaschisis ((Hovda et al. 1987; Sutton et al.2000). The prior art, however, further teaches that amphetamines do notimprove recovery following certain types of injury including lesions inthe substantia nigra (Mintz and Tomer 1986). The prior art also teachesthat administration of amphetamines (e.g., methamphetamine; MA) prior tofocal ischemia actually increases the infarct volume in cortical andstriatal regions (Wang et al. 2001).

A need still exist for a treatment that prevents neuronal damage beforeit occurs and actually provides neuronal protection after the occurrenceof a transient cerebral hypoxic and/or ischemic condition to minimize orprevent damage. Such a preventative method is disclosed herein, whichprovides a method of preventing or reducing damage to the cerebralneuronal cells before it occurs instead of trying to treat the damageafter occurrence and promote recovery.

SUMMARY OF THE INVENTION

The present invention is directed to a method of reducing the occurrenceof neuronal cell damage caused by transient cerebral hypoxia and/orischemia. The method preferably comprises the steps of: diagnosing asubject having a transient cerebral hypoxic and/or ischemic condition;and within 16 hours after onset of the condition, administering to thesubject a neuroprotective amount of a pharmaceutical agent. Thepharmaceutical agent is preferably selected from the group consistingof: a central nervous system stimulant (CNSS), monoamineneurotransmitter, monoamine oxidase inhibitor (MAOI), tricyclicantidepressant (TCA), or a combination thereof.

Preferred pharmaceutical agents includes amphetamines, methamphetamine,methylphenidate, methylenedioxymethamphetamine, or a combinationthereof.

In one specific embodiment, the pharmaceutical agent is methamphetamineadministered to the subject in unit dosage amounts of less than 5 mg/kg.

In other specific embodiments, the pharmaceutical agent is a combinationof methamphetamine, methylphenidate, methylenedioxymethamphetamine, or acombination thereof and at least one additional agent selected from thegroup consisting of: a monoamine neurotransmitter, MAOI, or a TCA. Theadditional agent can also include a monoamine neurotransmitter,preferably selected from the group consisting of: dopamine,norepinephrine, or serotonin.

The present invention preferably reduces the occurrence of cerebralneuronal cell damage, which includes cell death, and more preferably,reduces the occurrence of neuronal cell damage to the neuronal cells. Ina preferred embodiment, the present invention reduces the occurrence ofneuronal cell damage to the neuronal cells of the hippocampus.

Typically, the transient cerebral hypoxic and/or ischemic condition iscaused by loss of blood, a heart attack, strangulation, surgery (e.g.,cardiac surgery), a stroke, air-way blockage, ischemic optic neuropathy,spinal cord injuries, traumatic brain injury, or low blood pressure. Thecondition, however, can be caused by many conditions, conditions thatcause neuronal cell damage due to the lack of oxygen and/or glucosereaching the neuronal cells for a temporary period of time.

In certain preferred embodiments, the pharmaceutical agent isadministered within 16, 14, 12, 10, 8, 6, 4, or 2 hours after the onsetof the condition. The agent is preferably administered via a parenteralor oral route, but other routes are contemplated and can be useddepending on the condition.

In one embodiment, the pharmaceutical agent is administered in apharmaceutical composition comprising a pharmaceutically acceptablecarrier. The pharmaceutical composition can be an immediate or extendedrelease formulation depending on the condition and likelihood ofreoccurrence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: shows a neuroprotective dose response of MA followingoxygen-glucose deprivation (OGD). Representative images of propidiumiodide stained rat hippocampal slice cultures taken 48 hrs post-OGD areshown. Cultures were treated with the following doses of MA: (A) Non-OGDcontrol; (B) 125 μm MA added 5 min post-OGD; (C) 250 μm MA added 5 minpost-OGD; (D) 500 μm MA added 5 min post-OGD; (E) 1 mMμm MA added 5 minpost-OGD; (F) OGD only. Panal (G) shows statistical analysis of PIstaining reported as relative fluorescence intensity (IOD). **=p<0.01,One-way ANOVA, Dunnet's Post-Hoc (OGD), error bars=mean & SEM; (OGD)n=10, (Non-OGD) n=13, (1 mM MA) n=10, (500 μM MA) n=11, (250 μM MA) n=9,(125 μM MA) n=7

FIG. 2: shows a temporal analysis of MA mediated neuroprotectionfollowing OGD. Representative images of propidium iodide stained rathippocampal slice cultures taken 48 hrs post-OGD are shown. A 250 μMdose of MA was administered post-stroke at the time points indicated:(A) non-OGD; (B) 0-5 min post-OGD; (C) 2 hrs post-OGD; (D) 4 hrspost-OGD; (E) 8 hrs post-OGD; (F) 16 hrs post-OGD; (G) OGD-untreated.Panal (H) shows statistical analysis of PI staining reported as relativefluorescence intensity (TOD). n=4, *=p<0.05, One-way ANOVA, Dunnet'spost-hoc (OGD), error bars=mean & SEM

FIG. 3: shows the Mean (±SEM) distance traveled in a novel open fieldapparatus. Animals were tested 24 hrs following 5-min 2-VO (Isch) orsham surgery (Sham). Following surgery (1-2 min), gerbils receivedmethamphetamine (5 mg) or saline vehicle (0 mg). Gerbils were placed inthe center region and permitted to explore the novel environment for 5minutes and distance data were collected using an automated trackingsystem. Ischemic gerbils without methamphetamine treatment weresignificantly more active compared to the no drug sham group. Ischemicand sham gerbils treated with the drug were not different and drugtreatment failed to significantly alter activity levels relative to thecontrol condition. *P<0.05 vs. Isch+drug condition.

FIG. 4: shows individual histological rating scores of hippocampalsections evaluated 21 days after ischemic insult (Isch) or sham controlsurgery (Sham). Gerbils were treated with methamphetamine (5 mg) orvehicle (0 mg) 1-2 minutes following surgery. Damage to the hippocampalCA1 region was evaluated using a 4 point rating scale. A score of 0 (4-5compact layers of normal neuronal bodies), 1 (4-5 compact layers withpresence of some altered neurons), 2 (spares neuronal bodies with “ghostspaces” and/or glial cells between them), 3 (complete absence orpresence of only rare normal neuronal bodies with intense gliosis of theCA1 subfield) was assigned for each animal. Analysis revealed thattreatment with methamphetamine significantly reduced damage to thehippocampal CA1 following ischemic insult.

FIG. 5: are photomicrographs of hippocampal sections processed 21 daysafter ischemic insult or sham procedure followed by administration ofmethamphetamine (5 mg/kg) or vehicle. A 5-min 2-VO resulted in theselective loss of pyramidal neurons in the hippocampal CA1 region(Panels C, D). As expected, sham surgery (Panels A, B) did not result inany neuronal cell loss. Gerbils treated with methamphetamine 1-2 minutesfollowing ischemic insult failed to exhibit any damage to thehippocampus (Panels E, F). Sections were stained with cresyl violet.Scale bars=200 μm (A, C, E) and 60 μm (B, D, F).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention can be used to reduce the occurrence of cerebralneuronal cell damage, including cell death, caused by a transientcerebral hypoxic and/or ischemic condition. Preferably the methodreduces the occurrence of neuronal cell damage to the cells of thehippocampus. The transient cerebral hypoxic and/or ischemic conditioncan be caused by many conditions that cause lack of oxygen and/orglucose to the cerebral cells for a temporary period of time. Forexample, a heart attack, strangulation, surgery (e.g., cardiac surgery),a stroke, blood loss, air-way blockage, or low blood pressure.Preferably, the subject being treated is a mammal, e.g., monkey, dog,cat, horse, cow, sheep, pig, and more preferably the subject is a human.

In contrast to the prior art, the present method actually providesprotection and prevents damage to cerebral neuronal cells after theoccurrence of transient cerebral hypoxia and/or ischemia instead ofsimply promoting recovery after the neuronal cell damage has all readybe caused. To provide the greatest neuronal protection to the subject,the neuroprotective agent should be administered to the subject within16 hours after onset (e.g., 10, 8, 6, 4, 2 hours) of the transientcerebral hypoxic and/or ischemic condition. The neuroprotective agent ispreferably selected from the group consisting of: a central nervoussystem stimulant (CNSS), monoamine neurotransmitter, monoamine oxidaseinhibitor (MAOI), tricyclic antidepressant (TCA), or a combinationthereof.

In a more preferred embodiment, the neuroprotective agent isamphetamine, methamphetamine, methylphenidate,ethylenedioxymethamphetamine, or combinations thereof. In one preferredembodiment, the amphetamine is a compound containing a phenylethylamine.In certain embodiments, the phenylethylamine is a d-amphetamine, such asdextroamphetamine, for example, dextroamphetamine aspartate,dextroamphetamine sulfate, dextroamphetamine saccharate,methamphetamine, etc. Specific non-limiting examples include, ADREX,BIPHETAMINE, DESOXYN, DEXEDRINE, FERNDEX, ROBESE, SPANSULE, OXYDESS II,DEXTROSTAT.

In one embodiment, the pharmaceutical agent is administered in apharmaceutical composition comprising a pharmaceutically acceptablecarrier. The pharmaceutical composition can be an immediate or extendedrelease formulation depending on the condition and likelihood ofreoccurrence. The compositions can further include otherpharmaceutically active compounds including, for example, at least oneadditional agent selected from the group consisting of: a monoamineneurotransmitter, MAOI, or a TCA. The additional agent can also includea monoamine neurotransmitter, preferably selected from the groupconsisting of: dopamine, norepinephrine, or serotonin, and morepreferably norepinephrine.

Those skilled in the art will recognize various synthetic methodologiesthat may be employed to prepare non-toxic pharmaceutically acceptablecompositions comprising the neuroprotective agent.

Pharmaceutical compositions can be prepared in individual dosage forms.Consequently, pharmaceutical compositions and dosage forms of theinvention comprise the active ingredients disclosed herein. The notationof “the pharmaceutical agent” or “neuroprotective agent” signifies thecompounds of the invention described herein or salts thereof.Pharmaceutical compositions and dosage forms of the invention canfurther comprise a pharmaceutically acceptable carrier.

In one embodiment, the term “pharmaceutically acceptable” means approvedby a regulatory agency of the Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, and more particularly in humans. The term “carrier” refersto a diluent, adjuvant, excipient, or vehicle with which an activeingredient is administered. Such pharmaceutical carriers can be liquids,such as water and oils, including those of petroleum, animal, vegetableor synthetic origin, such as peanut oil, soybean oil, mineral oil,sesame oil and the like. The pharmaceutical carriers can be saline, gumacacia, gelatin, starch paste, talc, keratin, colloidal silica, urea,and the like. In addition, other excipients can be used.

Single unit dosage forms of the invention are suitable for oral, mucosal(e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g.,subcutaneous, intravenous, bolus injection, intramuscular, orintraarterial), or transdermal administration to a patient. Examples ofdosage forms include, but are not limited to: tablets; caplets;capsules, such as soft elastic gelatin capsules; cachets; troches;lozenges; dispersions; suppositories; ointments; cataplasms (poultices);pastes; powders; dressings; creams; plasters; solutions; patches;aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage formssuitable for oral or mucosal administration to a patient, includingsuspensions (e.g., aqueous or non-aqueous liquid suspensions,oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a patient; and sterile solids (e.g., crystalline or amorphous solids)that can be reconstituted to provide liquid dosage forms suitable forparenteral administration to a patient. The agent is preferablyadministered via a parenteral or oral route, but other routes arecontemplated as discussed in detail herein and largely depend on theischemic condition.

The composition, shape, and type of dosage forms of the invention willtypically vary depending on their route of administration and animalbeing treated. For example, a parenteral dosage form may contain smalleramounts of one or more of the active ingredients it comprises than anoral dosage form used to treat the same disease. These and other ways inwhich specific dosage forms encompassed by this invention will vary fromone another will be readily apparent to those skilled in the art. See,e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing,Easton Pa. (1990).

Typical pharmaceutical compositions and dosage forms comprise one ormore excipients. Suitable excipients are well known to those skilled inthe art of pharmacy, and non-limiting examples of suitable excipientsare provided herein. Whether a particular excipient is suitable forincorporation into a pharmaceutical composition or dosage form dependson a variety of factors well known in the art including, but not limitedto, the way in which the dosage form will be administered to a patient.For example, oral dosage forms such as tablets may contain excipientsnot suited for use in parenteral dosage forms. The suitability of aparticular excipient may also depend on the specific active ingredientsin the dosage form. For example, the decomposition of some activeingredients may be accelerated by some excipients such as lactose, orwhen exposed to water.

The invention further encompasses pharmaceutical compositions and dosageforms that comprise one or more compounds that reduce the rate by whichan active ingredient will decompose. Such compounds, which are referredto herein as “stabilizers,” include, but are not limited to,antioxidants such as ascorbic acid, pH buffers, or salt buffers.

For a particular condition or method of treatment, the dosage isdetermined empirically, using known methods, and will depend upon factssuch as the biological activity of the particular compound employed, themeans of administrations, the age, health and body weight of the host;the nature and extent of the symptoms; the frequency of treatment; theadministration of other therapies and the effect desired. Hereinafterare described various possible dosages and methods of administrationwith the understanding that the following are intended to beillustrative only. The actual dosages and method of administration ordelivery may be determined by one of skill in the art. For example, whenthe neuroprotective agent is methamphetamine administered to humans, theunit dosage amount is typically less than 5 mg/kg. Great dosages aregenerally toxic and should not typically be used.

Frequency of dosage may also vary depending on the compound used andwhether an extended release formulation is used. However, for treatmentof most disorders, a single dose is preferred.

Oral Dosage Forms

Pharmaceutical compositions of the invention that are suitable for oraladministration can be presented as discrete dosage forms, such as, butare not limited to, tablets (e.g., chewable tablets), caplets, capsules,and liquids (e.g., flavored syrups). Such dosage forms containpredetermined amounts of active ingredients, and may be prepared bymethods of pharmacy well known to those skilled in the art. Seegenerally, Remington's Pharmaceutical Sciences, 18th ed., MackPublishing, Easton Pa. (1990).

Typical oral dosage forms of the invention are prepared by combining theactive ingredients in an intimate admixture with at least one excipientaccording to conventional pharmaceutical compounding techniques.Excipients can take a wide variety of forms depending on the form ofpreparation desired for administration. For example, excipients suitablefor use in oral liquid or aerosol dosage forms include, but are notlimited to, water, glycols, oils, alcohols, flavoring agents,preservatives, and coloring agents. Examples of excipients suitable foruse in solid oral dosage forms (e.g., powders, tablets, capsules, andcaplets) include, but are not limited to, starches, sugars,micro-crystalline cellulose, diluents, granulating agents, lubricants,binders, and disintegrating agents.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit forms, in which case solidexcipients are employed. If desired, tablets can be coated by standardaqueous or nonaqueous techniques. Such dosage forms can be prepared byany of the methods of pharmacy. In general, pharmaceutical compositionsand dosage forms are prepared by uniformly and intimately admixing theactive ingredients with liquid carriers, finely divided solid carriers,or both, and then shaping the product into the desired presentation ifnecessary.

For example, a tablet can be prepared by compression or molding.Compressed tablets can be prepared by compressing in a suitable machinethe active ingredients in a free-flowing form such as powder orgranules, optionally mixed with an excipient. Molded tablets can be madeby molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

Examples of excipients that can be used in oral dosage forms of theinvention include, but are not limited to, binders, fillers,disintegrants, and lubricants. Binders suitable for use inpharmaceutical compositions and dosage forms include, but are notlimited to, corn starch, potato starch, or other starches, gelatin,Natural and synthetic gums such as acacia, sodium alginate, alginicacid, other alginates, powdered tragacanth, guar gum, cellulose and itsderivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethylcellulose calcium, sodium carboxymethyl cellulose), polyvinylpyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropylmethyl cellulose, (e.g., Nos. 2208, 2906, 2910), microcrystallinecellulose, and mixtures thereof.

Suitable forms of microcrystalline cellulose include, but are notlimited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICELRC-581, AVICEL-PH-105 (available from FMC Corporation, American ViscoseDivision, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof. Anspecific binder is a mixture of microcrystalline cellulose and sodiumcarboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or lowmoisture excipients or additives include AVICEL-PH-103 and Starch 1500LM.

Examples of fillers suitable for use in the pharmaceutical compositionsand dosage forms disclosed herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.The binder or filler in pharmaceutical compositions of the invention istypically present in from about 50 to about 99 weight percent of thepharmaceutical composition or dosage form.

Disintegrants are used in the compositions of the invention to providetablets that disintegrate when exposed to an aqueous environment.Tablets that contain too much disintegrant may disintegrate in storage,while those that contain too little may not disintegrate at a desiredrate or under the desired conditions. Thus, a sufficient amount ofdisintegrant that is neither too much nor too little to detrimentallyalter the release of the active ingredients should be used to form solidoral dosage forms of the invention. The amount of disintegrant usedvaries based upon the type of formulation, and is readily discernible tothose of ordinary skill in the art. Typical pharmaceutical compositionscomprise from about 0.5 to about 15 weight percent of disintegrant,preferably from about 1 to about 5 weight percent of disintegrant.

Disintegrants that can be used in pharmaceutical compositions and dosageforms of the invention include, but are not limited to, agar-agar,alginic acid, calcium carbonate, microcrystalline cellulose,croscarmellose sodium, crospovidone, polacrilin potassium, sodium starchglycolate, potato or tapioca starch, other starches, pre-gelatinizedstarch, other starches, clays, other algins, other celluloses, gums, andmixtures thereof.

Lubricants that can be used in pharmaceutical compositions and dosageforms of the invention include, but are not limited to, calciumstearate, magnesium stearate, mineral oil, light mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, andmixtures thereof. Additional lubricants include, for example, a syloidsilica gel (AEROSIL 200, manufactured by W.R. Grace Co. of Baltimore,Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co.of Plano, Tex.), CAB-O-SIL (a pyrogenic silicon dioxide product sold byCabot Co. of Boston, Mass.), and mixtures thereof. If used at all,lubricants are typically used in an amount of less than about 1 weightpercent of the pharmaceutical compositions or dosage forms into whichthey are incorporated.

A preferred solid oral dosage form of the invention comprises an activeingredient, anhydrous lactose, microcrystalline cellulose,polyvinylpyrrolidone, stearic acid, colloidal anhydrous silica, andgelatin.

Parenteral Dosage Forms

Parenteral dosage forms can be administered to patients by variousroutes including, but not limited to, subcutaneous, intravenous, bolusinjection, intramuscular, and intraarterial. Because theiradministration typically bypasses patients' Natural defenses againstcontaminants, parenteral dosage forms are preferably sterile or capableof being sterilized prior to administration to a patient. Examples ofparenteral dosage forms include, but are not limited to, solutions readyfor injection, dry products ready to be dissolved or suspended in apharmaceutically acceptable vehicle for injection, suspensions ready forinjection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage forms ofthe invention are well known to those skilled in the art. Examplesinclude, but are not limited to: Water for Injection USP; aqueousvehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water-miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

The present invention will now be illustrated by the followingnon-limiting examples. It is to be understood that the foregoingdescribes preferred embodiments of the present invention and thatmodifications may be made therein without departing from the spirit orscope of the present invention as set forth in the claims.

Examples

The neuroprotective efficacy of amphetamines following transientcerebral ischemic insult was not previously investigated. In the presentstudy, methamphetamine (MA) was evaluated using in vitro and in vivomodels of transient cerebral ischemia. For the in vitro model, rathippocampal slice cultures were challenged with oxygen-glucosedeprivation. In a second series of experiments, a 5-min 2-VO occlusiongerbil model was used in combination with behavioral testing to test theneuroprotective efficacy of MA in vivo. During the present study it wassurprisingly discovered and demonstrated that MA administration within16 hours following transient cerebral ischemia is actuallyneuroprotective, reducing neuronal cell damage, including death.

Materials and Methods

1.1 Animals

All experimental animal procedures were approved by the UniversityInstitutional Animal Care and Use Committee. Twenty-eight adult maleMongolian gerbils (Meriones unguiculatus) weighing 60-80 gm were usedfor the in vivo experiments. These animals were housed individually in alight- (12 h light/dark cycle) and temperature- (23° C.) controlledenvironment. Commercial rodent pellets and water were provided adlibitum.

1.2 In Vitro Hippocampal Slice Studies

Neonatal rats (Sprague-Dawley) at postnatal Day 7 (P7) were decapitatedand the hippocampi dissected out under sterile conditions. Thehippocampi were chopped into 400 μm slices on a McIlwain tissue chopperand individual slices were cultured on Millicell permeable membranes(0.4 μM pore size) in six well plates for 6 days at 37° C. in 5% CO2.For the first two days, the slices were maintained in a primary platingmedia (50% DMEM (+) glucose, 25% HBSS (+) glucose, 25% heat inactivatedhorse serum, 5 mg/mL D-glucose (Sigma), 1 mM Glutamax, 1.5%PenStrep/Fungizone (Gibco), and 5 mL of 50×B27 (Gibco) supplement plusanti-oxidants that was changed every 24 h. On the fourth day, the sliceswere placed in serum-free neurobasal medium (10 mL Neurobasal-A, 200 μLof 50×B27 supplement, 100 μL of 100× Fungizone, and 100 μL of 100×Glutamax) and this media was changed every 48 hrs. 24 hrs prior toexperimentation, the inserts were placed in a serum-free neurobasalmedia and B27 supplement without antioxidants. Prior to theoxygen-glucose deprivation (OGD), a glucose free balanced salt solution(BSS) (120 mM NACl, 5 mM KCl, 1.25 mM NaH2PO4, 2 mM MgSO4, 2 mM CaCl2,25 mM NaHCO3, 20 mM HEPES, 25 mM sucrose pH of 7.3) was infused for 1hour with 5% CO2 and 10 L/hr nitrogen gas. The inserts were thentransferred into deoxygenated BSS and placed in a 37° tank (Pro-Ox) withan oxygen feedback sensor that maintained gas levels at 0.1% O2, 5% CO2,94.4% Nitrogen for 90 m. After OGD, the slices were immediatelytransferred back into prewarmed neurobasal media and assayed perexperimental protocols.

1.3 Transient Cerebral Ischemia

Gerbils were anesthetized with isoflurane and core-body temperaturemaintained at 37-38° C. during surgery using a homeothermic blanket(Harvard Apparatus, South Natick, USA). A midline incision was made inthe neck and the common carotid arteries were isolated and occluded for5 min using 85-gm pressure aneurysm clips (ISCH; n=14). A second groupof gerbils (SHAM; n=14) underwent the identical procedure except thecarotid arteries were not clamped. The incision was sutured and animalsreceived MA (5 mg/kg; i.p) or equal volume of vehicle (saline; 0 mg)within 2 minutes of reperfusion. Animals were placed in a warmed cage,and observed for 30 minutes. Tylenol (8 mg/ml) was added to drinkingwater to provide postoperative analgesia.

1.4 Behavioral Testing and Histological Evaluation.

Each gerbil was tested 48 hrs following surgery in an open-fieldapparatus consisting of a metal screen floor 77 cm×77 cm with walls 15cm in height. Animals were placed in the center region and permitted toexplore the novel environment for 5 minutes. Behavioral data (distancetraveled, speed) were collected using an automated tracking system(ANY-maze, Stoelting, Ill.) and evaluated separately using ANOVA and theappropriate post hoc test (P<0.05 considered significant). Twenty-onedays post-surgery, gerbils were euthanized with CO2 and perfused withphosphate buffered saline followed by 4% paraformaldehyde. Tissue fromsham gerbils treated with MA (SHAM+0 mg) was not evaluated since acuteadministration of MA was not expected to histologically alter thehippocampus of this group. Brains were removed and post-fixed for atleast 48 hrs prior to collection of 40 μm vibratome sections through thehippocampal region. Sections were mounted on slides and stained withcresyl violet. Damage to the hippocampal CA1 region was evaluatedwithout knowledge of treatment condition by two independent observersusing a 4 point rating scale described elsewhere (Babcock et al. 1993).A score of 0 (4-5 compact layers of normal neuronal bodies), 1 (4-5compact layers with presence of some altered neurons), 2 (sparesneuronal bodies with “ghost spaces” and/or glial cells between them), 3(complete absence or presence of only rare normal neuronal bodies withintense gliosis of the CA1 subfield) was assigned for each animal.Ratings were averaged and evaluated using nonparametric statistics(Kruskal-Wallis and Mann-Whitney U test; P<0.05 considered significant).

Results

2.1 In Vitro Hippocampal Slice Studies

Hippocampal rat slices exposed to 90 min of oxygen glucose deprivation(OGD) and treated with methamphetamine (MA) showed significantly(p=<0.01) decreased levels of propidium iodide (PI) uptake indicatingdecreased neuronal death when compared to OGD only slices (FIG. 1). Indose response studies with MA, we observed optimal dosing with 250 μM MAand increasing PI uptake as the concentration increased or decreasedfrom this amount. However, at all concentrations tested (125 μM, 250 μM,500 μM, 1 mM) we observed significant neuroprotection (p=<0.01) whencompared to OGD-only slices.

To further elucidate the effect of MA we added 250 μM at various timepoints after OGD and found that MA significantly (p=<0.05) decreasedneuronal death when administered up to 16 hrs after OGD. Addition of MA24 hrs post-OGD decreased neuronal death but did not significantlydiffer from OGD.

2.2 Transient Cerebral Ischemia Studies

Gerbils exhibited coordinated movements within 10 minutes of isofloranetermination. Animals treated with MA became piloerect with their tailspointing up. Animals were tested in an open field apparatus 48 hrsfollowing surgery. Gerbils that underwent ischemic insult without MAtreatment traveled 129.4 m (±20; SEM), while sham controls with andwithout drug treatment traveled 72.7 m (±6) and 73.2 m (±7.5),respectively. Ischemic gerbils treated with MA following surgerytraveled 66.3 m±5.6. Analysis of activity data revealed a significantinteraction between drug treatment and surgical conditions (P<0.05).Subsequent planned comparisons indicated that ischemic gerbils, in theabsence of MA treatment, were significantly more active compared to theno drug sham group (P<0.05). Ischemic and sham gerbils treated with MAwere not significantly different (P>0.05). Finally, treatment with MAfailed to significantly alter activity levels relative to the controlcondition (SHAM+0 mg vs. SHAM+5 mg; P>0.05). Analysis of speed data(distance traveled/time) revealed a similar pattern with ischemicgerbils treated with saline (ISCH exhibiting significantly fastestspeeds relative to all other experimental groups (data not shown).

The histopathology scores and representative photomicrographs of theevaluated groups are illustrated in FIGS. 3 and 4, respectively. Gerbilsin the ISCH+0 mg condition exhibited extensive damage to the hippocampalCA1 region. Four of six gerbils in this group had complete absence ofnormal neuronal bodies with intense gliosis of the CA1 subfield. Incontrast, all of the gerbils in the SHAM+0 mg group were rated as havingno detectable damage to the hippocampus (mean rating 0±0). Six of theanimals in the ISCH+5 mg MA group exhibited 4-5 compact layers of normalneuronal bodies in the hippocampus (group rating 0.07±0.07). Only 1gerbil in this condition exhibited any detectable damage to the CA1region. Analysis of rating scores revealed a significant differencebetween groups (P<0.05).

Subsequent evaluation of individual group data indicated that SHAM+0 mgand ISCH+5 mg conditions were not significantly different (P>0.05) andboth of these conditions were significantly different from the ISCH+0 mggroup (P<0.05).

Discussion

The results of the present study indicate that if a neuroprotectiveagent, e.g., MA, is administered within 16 hours after transientischemic insult, damage to the neuronal cells may be reduced orprevented in the hippocampus. MA, for example, resulted in adose-dependent neuroprotective response in rat hippocampal slicecultures challenged with oxygen-glucose deprivation. The 250 μM doseshowed the greatest degree of protection and was effective whenadministered up to 16 hours following oxygen-glucose deprivation. At 24hrs post-OGD MA administration did not significantly reduce PI uptakeindicating that MA dosing must occur within a relatively short timeperiod after OGD to activate the mechanism(s) responsible for reducingneuronal damage and death.

The neuroprotective efficacy of MA was also demonstrated in vivo using a5-min gerbil 2-VO transient ischemia model. MA administration within 1-2minutes of reperfusion prevented any significant loss of hippocampal CA1pyramidal cells. The histological evaluation revealed that ischemicgerbils treated with MA exhibiting almost complete protection of thehippocampal CA1 region with only 1 of 7 animals exhibited any detectableneuronal pathology in the hippocampus. A 5-min bilateral carotidocclusion in the gerbil produces increased locomotor activity thatcorrelates with hippocampal CA1 cell death (Wang and Corbett 1990;Babcock et al. 1993). The locomotor activity of ischemic gerbils treatedwith MA in the present study was comparable to control levels, which isindicative of significant neuroprotection. It is entirely possible thatthe arousal and hyperactivity that amphetamines produce could interactwith the behavioral effects of ischemia. However, behavioral testing inthe present study was conducted after the drug should have beenmetabolized (48 hrs). Consistent with this interpretation was theobservation that control gerbils treated with MA were not hyperactiverelative to animals that received saline (SHAM+0 mg). The dose of MAused in the in vivo experiment was derived from a previous report thatused gerbils (Teuchert-Noodt et al. 2000; Araki et al. 2002) as anexperimental model. We also conducted a preliminary study in which dosesof MA greater than 5 mg/kg (e.g., 10 and 20 mg/kg) were found to belethal in gerbils following surgery and were not evaluated further.

Amphetamine in combination with training has been shown to be apromising pharmacological strategies for behavioral recovery from stroke(see Martinsson and Eksborg, 2004). Our observation that MA actuallyprevents detectable hippocampal damage following ischemic insult ifgiven within a particular time frame after insult, i.e., within 16hours, represents a novel finding. It is notable that these findingsshow that neuroprotection is independent of any behavioral trainingfollowing the insult. It is possible that the ability of MA to actuallyprotect and prevent the hippocampus from neuronal damage, in contrast tothe prior art teachings of treatment after damage has occurred, iseffect in with transient cerebral ischemia. Unlike focal ischemia orother types of cortical injury, transient cerebral ischemia ischaracterized by a pattern of delayed cell death limited to hippocampalpyramidal cells. The reperfusion that follows the brief ischemic episodein this model is a key event for the subsequent cell death that occurs3-5 days following insult.

Current studies of MA administration prior to an acute stroke eventindicate that MA significantly increases neuronal death (Wang et al.2001). However, in light of our current findings, it is entirelypossible that treatment with MA prior to a stroke event depletes storesof dopamine and norepinephrine that remain unavailable for release aftera stroke event, and the subsequent decrease in neuronal signaling may beplaying a key role in the damage observed in MA pre-treatment andstroke. The ability of CNSS, e.g., MA, to induce an extremely largerelease of these neurotransmitters in a very short time span maypartially explain the neuroprotective effect we observed in ourexperiments. Future research aimed at understanding the neuroprotectivemechanism of CNSS agents may further elucidate the exact mechanism andtreatment for acute ischemic events.

The preceding technological disclosure describes illustrativeembodiments of the method of reducing the occurrence of neuronal celldamage caused by transient cerebral hypoxia and/or ischemia and is notintended to limit the present invention to these precise embodiments.Further, any changes and/or modifications, which may be obvious by onewith ordinary skill in the related art, including but not limited topharmaceutical salt derivatives or non-functional changes are intendedto be included within the scope of the invention.

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1-20. (canceled)
 21. A method of reducing the occurrence of brain celldamage or death caused by transient cerebral hypoxia and/or ischemia,the method comprising the steps of: diagnosing a subject having atransient cerebral hypoxic and/or ischemic condition; and within 16hours after onset of the condition, administering to the subject atherapeutic effective amount of methamphetamine.
 22. The method of claim21, wherein the methamphetamine administered to the subject in unitdosage amounts of less than 5 mg/kg.
 23. The method of claim 21, whereintreatment reduces the occurrence of neuronal cell damage to brain cellsof the hippocampus.
 24. The method of claim 21, wherein the condition iscaused by low blood pressure, blood loss, a heart attack, traumaticbrain injury, strangulation, surgery, a stroke, ischemic opticneuropathy, or air-way blockage.
 25. The method of claim 24, wherein inthe condition is caused by cardiac surgery.
 26. The method of claim 24,wherein the condition is caused by traumatic brain injury.
 27. Themethod of claim 21, wherein administration occurs within 12 hours afteronset of the condition and only a single dose of the methamphetamine isadministered.
 28. The method of claim 21, wherein, the administering isvia a bolus injection.
 29. The method of claim 28, wherein themethamphetamine is in a pharmaceutical composition comprising apharmaceutically acceptable carrier.
 30. The method of claim 29, whereinthe pharmaceutical composition is an extended release formulation. 31.The method of claim 21, wherein the subject is a human in need of suchtreatment.
 32. The method of claim 31, wherein the condition is causedby an ischemic stroke, cardiac surgery or ischemic optic neuropathy. 33.The method of claim 31, wherein the methamphetamine is administeredwithin 12 hours of surgery.
 34. The method of claim 31, wherein themethamphetamine is administered within 2 hours of surgery.
 35. A methodof reducing the occurrence of brain cell damage or death caused bytraumatic brain injury, the method comprising the steps of: diagnosing asubject having traumatic brain injury; and within 16 hours after onsetof the injury, administering to the subject a therapeutic effectiveamount of methamphetamine.
 36. The method of claim 35, wherein, theadministering is via a bolus injection.
 37. The method of claim 35,wherein the methamphetamine is in a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier.
 38. The method ofclaim 35, wherein the subject is a human.